Protocol for phospho-SrcKD: rPTPεD1 complex preparation and BLI binding assays to demonstrate their exosite interface

Summary Here, we present a protocol for the in vitro phosphorylation of Src kinase domain (SrcKD), preparation of phospho-SrcKD in complex with the D1 domain of rPTP epsilon (rPTPεD1), and binding assays using biolayer interferometry (BLI). We describe steps for the in vitro phosphorylation of SrcKD and preparation of the phospho-SrcKD: rPTPεD1 complex for small-angle X-ray scattering (SAXS) experiments. We then detail instructions for the BLI binding assay to determine the binding affinity between phospho-SrcKD and rPTPεD1. For complete details on the use and execution of this protocol, please refer to EswarKumar et al.1


BEFORE YOU BEGIN
To analyze the function and dynamics of phospho-protein: protein complexes, it is crucial to understand their structures. 2However, the phospho-protein: protein interactions involved in the signaling pathway make this difficult as they are highly transient and prone to easy dissociation, 3 and their complex preparation at the milligram scale for structure determination is challenging.The current protocols detail the steps needed to purify the phospho-SrcKD: rPTPεD1 complex and perform BLI binding assays to validate the identified interface.They follow those published in EswarKumar et al. 1,4 In outline, Src-KD, rPTPεD1 & C-terminal Src kinase (CSK) genes are cloned and expressed in a bacteria expression system.The proteins are then purified using immobilized metal affinity and size exclusion chromatography (please refer to EswarKumar et al. for a detailed protocol 1 ).First, the phosphorylated form of SrcKD is prepared, which allows us to purify the phospho-SrcKD: rPTPεD1 complex by size-exclusion chromatography for SAXS experiments.For the binding assays, phosphorylated SrcKD and rPTPεD1 are purified separately, and the association rates are determined using BLI.

STEP-BY-STEP METHOD DETAILS
In vitro phosphorylation of SrcKD To determine the structure of the phospho-SrcKD: rPTPεD1 complex and to demonstrate the mechanism of their interaction, phospho-SrcKD and rPTPεD1 are produced separately.It is already established that CSK phosphorylates SrcKD. 5This section describes SrcKD phosphorylation by CSK in the presence of ATP, MnCl 2 , and MgCl 2 (Figure 1).Note: Before use, the column should be pre-equilibrated with SEC buffer (All the buffers used in this protocol are listed in Table 1).3. Concentrate the purified SrcKD and CSK-His-CBD to 60 mM and 2 mM, respectively for phosphorylation.4. To achieve the phosphorylation of SrcKD, add 10 mM MgCl 2 , 10 mM MnCl 2 , 10 mM DTT, 120 mM ATP, 2 mM CSK-His-CBD and 5% glycerol in 1 mL of SrcKD solution with a final concentration of 58 mM. 5. Incubate the reaction mixture at 25 C for 30 min.6. Next, use chitin beads to get rid of the CSK-His-CBD from the reaction mixture containing phosphorylated SrcKD and CSK-His-CBD.
Note: Pre-equilibrated the chitin beads with 20 mM Tris pH 8.0, 50 mM NaCl, 10 mM MgCl 2 and 5 mM DTT 7. Now, the flow-through contains phosphorylated SrcKD for complex formation with rPTPεD1.
Note: At least 5 mM DTT needs to be added during the process.We observed SrcKD precipitation with less than 5 mM DTT.
Purification of the phospho-SrcKD: rPTPεD1 complex Timing: 2 h 8. Mix the phosphorylated SrcKD (phospho-SrcKD) with 0.5 M excess of purified rPTPεD1 and incubate for 10-30 min at 25 C (Figure 2A).9. Separate the phospho-SrcKD: rPTPεD1 complex from un-complexed proteins with the Superdex 200 Increase 10/300 column at 4 C in the presence of SEC buffer using the AKTA purifier.
Note: Pre-equilibrate the column with SEC buffer prior to the experiment.
10. Inject 500 mL of sample volume into the AKTA purifier at a flow rate of 0.5 mL per minute.11.Confirm the phospho-SrcKD: rPTPεD1 complex formation by elution volume, SDS-PAGE electrophoresis, and analytical ultra-centrifugation (AUC).a.The molecular weights of SrcKD and rPTPεD1 are similar, so the un-complexed form of unphosphorylated SrcKD and rPTPεD1 elutes at 15.6 mL, whereas the phospho-SrcKD: rPTPεD1 complex elutes at 14.7 mL.b.Almost a 1 mL peak shift between the complex and individual proteins in SEC is required based on their molecular weights, as shown in Figure 2B.c.SDS-PAGE results show two corresponding bands representing the rPTPεD1: phospho-SrcKD complex (Figure 2C).d.The AUC results should be consistent with SEC results.The individual SrcKD and rPTPεD1 show a single peak with an S 20 of 3, whereas the mixture of phospho-SrcKD: rPTPεD1 shows an additional peak with S 20 value > 4 (Figure 2D).
Note: Using no more than 50 mM NaCl in the buffer is crucial to the preservation of the rPTPεD1: SrcKD complex.The presence of 100 or 150 mM NaCl in the buffer causes the complex to dissociate.

Protocol
The binding assays by biolayer interferometry Timing: 4 h 12.To perform the binding experiments, the rPTPεD1, phospho-His-SrcKD, and the variants (Table 1) need to be purified.

Note:
The rPTPεD1 and its variants are untagged.The protein variants are listed in the key resources table.
13.The Ni-NTA biosensor tips on the BLItz system by ForteBio Inc. are used to measure the binding kinetics between rPTPεD1 and phospho-His-SrcKD.14.The experiments can be divided into four stages: a. Baseline measurements with BLI kinetic buffer (steps 1, 2), b.Loading phospho-His-SrcKD (or variants) onto the Ni-NTA biosensor (steps 3 to 5), c.Association with the binding partner, rPTPεD1 (or its variants) (step 6).d.Dissociation of the binding partner (step 7) (Figure 3).15.First, pre-hydrate the Ni-NTA sensors for 10 min in the BLI kinetics buffer.16.After hydration, attach the Ni-NTA biosensor to the BLI system and initialize baseline measurements for 30 s. 17.Load 5 mL of the phospho-His-SrcKD (at a concentration of 58 mM) as bait into the BLI drop holder and immobilize for 3 min on the Ni-NTA sensor tips.18. Wash the immobilized phospho-His-SrcKD with 250 mL of the BLI kinetics buffer loaded into the system before the BLI signal was measured for 90 s to prepare the biosensor for interaction with its partner.
Note: To maintain a stable phosphorylation level of His-SrcKD, supplement the buffer with a fresh 100 mM ATP and 1 mM CSK before every immobilization.
19. Once the bait protein reaches saturation, load 5 mL of the analyte (rPTPεD1 or its variants) into the BLI drop holder.Lower the arm to initiate association and measure the BLI signal for 120 s. 20.Later, add 250 mL of BLI kinetic buffer into the 0.5 mL microfuge tube and lower the biosensor to initiate dissociation and measure the BLI signal for 120 s. 21.After the final measurements, process the sensorgram using the BLItz Pro software (ForteBio Inc.).22.For data analysis, select global fitting and non-linear regression analysis.Examine the fitting results by observing the overlay of regression curves on sensor data traces.
Note: For reference, a table with determined parameter values such as rate constants, R max , K A , K D (Table 1) and statistics like the standard error of parameters and Chi-squared is outputted.
23. Lastly, export the data to text files and generate publication-quality sensorgrams using GraphPad Prism version 7 software 7,8 Note: The hydrolysis of phosphorylation on protein is a danger that needs to be avoided.Maintaining phosphorylation is critical to obtaining reliable binding constants.Fresh ATP solution and stable CSK should be supplemented before every immobilization.All steps in binding experiments should be carried out promptly and any waiting time should be avoided.

EXPECTED OUTCOMES
By following this protocol, recombinant SrcKD and rPTPεD1 can be purified and phosphorylated SrcKD obtained in around 2 weeks.The phospho-SrcKD: rPTPεD1 complex at mg scale can be prepared for structural studies, and binding constants between phospho-Src and rPTPεD1 can be determined.This protocol will be useful to researchers studying phospho-protein: protein interactions.

LIMITATIONS
Biolayer interferometry is a label-free technology to measure biomolecular interaction directly.The binding between the protein immobilized on the biosensor tip surface and the binding partner in the solution increases the optical thickness at the biosensor tip, resulting in a wavelength shift.However, any non-specific binding between the biosensor tip and the binding partner can also yield a wavelength shift.Although BLItz has a relatively short machine dead time and requires as little as 4 mL of the analyte, it is a less sensitive and low throughput instrument.

TROUBLESHOOTING Problem 1 [
Step 8] During complex preparation, the phospho-SrcKD: rPTPεD1 complex dissociates due to its transient nature in the presence of 100 mM or more of NaCl.

Potential solution
Various NaCl concentrations from 50-150 mM can be screened.Our results show that the phospho-SrcKD: rPTPεD1 complex remains stable at 50 mM NaCl, enabling its purification through SEC experiments.
Problem 2 Step 12] Different instruments, such as Surface Plasmon Resonance (SPR), isothermal calorimetry (ITC), and microscale thermophoresis (MST) are used to measure the binding affinities between the phospho-SrcKD and rPTPεD1 but fail to obtain accurate binding constants due to phospho-tyrosine hydrolysis on phospho-SrcKD during the long instrument dead time unavoidable with their use.

Potential solution
Ultimately, the BLItz system with a short dead time may need to be used.In addition, ATP and CSK can be supplemented before every binding measurement, which overcame the problem in our lab.

RESOURCE AVAILABILITY
Lead contact Further information and reasonable requests for reagents may be directed to, and will be fulfilled by the lead contact, Meng-Chiao Ho (joeho@gate.sinica.edu.tw).

Technical contact
Questions about the technical specifics of this protocol should be directed to the technical contact, Meng-Chiao Ho (joeho@gate.sinica.edu.tw).

Materials availability
All unique reagents generated in this study are available upon reasonable request from the lead contact.

1 .
First, clone SrcKD with an N-terminal His tag (His-SrcKD), and CSK with a His tag and a chitin-binding domain (CBD) at the C-terminus (CSK-His-CBD).2. Use immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC) to purify both His-SrcKD and CSK-His-CBD.a. Collect the pure fractions containing His-SrcKD, pool them together, and treat with His-TEV protease (1 mg of TEV per 50 mg of fusion protein) to remove the His tag. 6b.Purify the resulting tag-free SrcKD, digested His-tag, and TEV protease proteins further with a second IMAC.c. Concentrate the flow-through from the second IMAC containing SrcKD and further purify using the HiLoad Superdex 200 16/600 SEC column.

Figure 1 .
Figure 1.Protocol for in vitro phosphorylation SrcKD (A) Step 1: Preparation of reaction mixture for in vitro SrcKD phosphorylation.(B) Step 2: Incubation of the reaction mixture at room temperature for 30 min.(C) Step 3: Separation of phospho-SrcKD from the mixture using chitin beads trap.

Figure 2 .
Figure 2. Purification of phospho-SrcKD:rPTPεD1 complex (A) Illustration of in vitro phosphorylation of SrcKD and preparation of the phosphor-SrcKD: rPTPεD1 complex.(B)An overlaid SEC profile of the phospho-SrcKD: rPTPεD1 complex colored in violet and a mixture of rPTPεD1 and SrcKD (un-complex form) colored in green.The elution positions of complex and un-complexed proteins are indicated as red and black arrows, respectively.The SEC was performed using the Superdex 200 10/300 GL column.(C) The SDS-PAGE result from the SEC of the phospho-SrcKD: rPTPεD1 complex.The first lane is the reference marker with the corresponding MW shown.Lanes 2-4 correspond to the highest peak area and lanes 5-8 correspond to the second peak area from the SEC (D) Distribution of the sedimentation coefficients of the rPTPεD1, phospho-SrcKD, and phospho-SrcKD: rPTPεD1 complex are shown in blue, yellow and violet, respectively.The complex revealed an additional peak with a S 20,w value of 4.11.

Figure 3 .
Figure 3. Illustration of the binding affinity between phosphor-His-SrcKD and rPTPεD1 by BLITz (A) Illustration of the detailed BLI binding protocol.(B) Example Blitz sensorgrams of various concentrations of rPTPεD1 binding to immobilized phospho-His-SrcKD.

Table 1 .
Affinities (K D ) & association/dissociation parameters (k on/off ) of the wild type & variants